SllmmaryThe CD40 surface membrane molecule plays an important role in the activation of mature human B cells, but its role in earlier stages orB lineage development is unknown. Here, we have investigated the effects of triggering the CD40 antigen on B cell precursors (BCP) by crosslinking with anti-CD40 antibody presented by Fc'/-receptor type II-transfected murine Ltk-cells (CD40 system). CD10 + surface immunoglobulin negative (slg-) BCP, freshly isolated from fetal bone marrow or precultured on stromal cells, proliferated in the CD40 system. This effect required the presence of IL-3, which acted as a specific cosignal among a panel of cytokines examined. The association of IL-10 and IL-7 potentiated the observed IL-3 and CD40-dependent BCP proliferation, demonstrating that IL-10 can act on early B lineage cells. CD40-dependent activation of fetal BCP did not favor maturation to slg + B cells, but resulted in the induction of high levels of surface membrane CD23. The emerging CD23 + BCP lacked slg and CD10, and represented an important proportion of the cycling cells in the CD40-dependent cultures. Taken together, our data demonstrate that stimulation of the CD40 antigen induces expression of the CD23 gene, and regulates cell proliferation during normal human B cell ontogeny.T he life history orB lymphocytes can be divided into two major steps. First, an antigen-independent process (B lymphopoiesis) permits constant generation of new immunocompetent surface (s)llgM + slgD + mature B cells in the bone marrow (for reviews see references 1 and 2). Then, mature B cells can be driven by antigens to proliferate and differentiate in secondary lymphoid organs (immunopoiesis) (for a review see reference 3). Both soluble cytokines and cell membrane-associated molecules regulate the various steps of B lymphocyte development. In this context, the CD40 B cell surface membrane molecule has recently been identified as playing a fundamental role in the process of immunopoiesis. Human CD40 is a 50-kD glycosylated phosphoprotein (4), that belongs to a new superfamily of receptors that includes the receptors for nerve growth factor and TNF (5, 6).Anti-CD40 antibody presented by Fc',/receptor type II (Fc'yRII) (CDw32)-transfected murine fibroblastic Ltkcells (CD40 system) induces strong and long-lasting B cell expansion in the presence of IL-4 (7, 8). The proliferation of anti-CD40-activated mature B cells is also enhanced by IL-10 (9). More strikingly, IL-10 allows CD40-activated B cells to produce large quantities of IgM, IgG, and IgA after their differentiation into plasma cells. Culturing CD40-activated naive slgD + slgM + B cells in the presence of IL-4 or IL-10 and TGF-~ induces the production of IgE or IgA, respectively, as a consequence of isotype switching (10-14).Recent studies have indicated that CD40 crosslinking represents the first step of the interaction between T and B cells. A ligand to CD40 has recently been identified and expressed from activated T cells, and CD40-Ig fusion proteins inhibit T cell-dependent B ce...
A novel kappa protein, encoded by a germline JC kappa transcript, is expressed by normal and leukemic human B cell precursors. The transcript displays an open reading frame initiated by a non‐AUG codon, and predicts a 15 kDa molecule which could be readily confirmed by in vitro translation. Cellular expression was demonstrated by immunofluorescence, precipitation and Western blotting. Furthermore, 2‐D gel electrophoresis revealed that germline JC kappa can covalently associate with mu heavy chain at the surface of pre‐B cells. We therefore propose that during B cell lymphopoiesis, two alternative pathways could be operative in which mu heavy chain can either associate with lambda 5 or germ‐line JC kappa.
Growth of human B-cell precursors (BCP) was achieved by plating fetal CD10+ surface-mu (s mu)- cells in liquid medium onto bone marrow- derived fibroblastic stromal cell layers deprived of hematopoietic cells. Proliferation of the fetal BCP was strongly potentiated by the addition of interleukin-7 (IL-7) to the cultures. Cultures included both a stroma-adherent and -nonadherent fraction of lymphoid cells, allowing us to expand the number of input BCP to 13-fold. In the presence of exogenous IL-7, proliferation was dose-dependent relative to the number of stromal cells, demonstrating that soluble IL-7 does not act alone to promote optimal growth. We further showed that the lymphoid cells recovered remain CD10+ sIg- BCP and that most cells expressed the maturation-associated CD20 antigen when IL-7 was added to the cultures. Whereas both freshly isolated CD20- and CD20bright BCP proliferated in the presence of stroma, we observed that high- proliferative capacity CD20dim cells were maintained in the cultures. Finally, CD20dim sorted cells were shown to subsequently acquire high levels of CD20 expression in culture, thus demonstrating a partial maturation sequence. The present culture system thus represents a useful model for studying the regulatory signals in early human B lymphopoiesis.
Anti-CD3-activated human CD4+ T cell clones were found to induce proliferation of CD10+, CD19+, surface(s) Ig- B cell precursors (BCP) isolated from human fetal bone marrow. The great majority of the B lineage cells recovered in cocultures of BCP and activated T cells displayed a BCP phenotype (Ig- or cytoplasmic mu+ and kappa/lambda-), including most of the cycling cells, indicating that the cultures do not favor a transition to mature B cells. Supernatants of activated T cells were ineffective in inducing BCP proliferation, indicating the necessity of close association with stimulator cells. In line with this finding, the CD40 molecule was found to represent an important component of the cocultures, as BCP proliferation was strongly inhibited by soluble anti-CD40 antibody. In addition, CD4+ T cell clones from a hyper-IgM patient expressing a truncated CD40 ligand (CD40-L) failed to induce BCP proliferation. Finally, a combination of cytokines (IL-2, IL-3, IL-7, and IL-10) enhanced the observed T cell-dependent BCP proliferation, but could not substitute for the deficient CD40-L. Taken together, our data demonstrate that CD4+ T cells exert a stimulatory effect on in vitro B human lymphopoiesis via the CD40 pathway. The present results suggest that T cells may play an important role in regulating B cell ontogeny in the bone marrow.
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